A fixed-duration, measurable residual disease-guided approach in CLL: follow-up data from the phase 2 ICLL-07 FILO trial.

Trials assessing first-line, fixed-duration approaches in chronic lymphocytic leukemia (CLL) are yielding promising activity, but few long-term data are available. We report follow-up data from a phase 2 trial (ICLL07 FILO) in previously untreated, medically fit patients (N = 135). Patients underwent obinutuzumab-ibrutinib induction for 9 months; then, following evaluation (N = 130 evaluable), those in complete remission and with bone marrow measurable residual disease (BM MRD) <0.01% (n = 10) received ibrutinib for 6 additional months; those in partial remission and/or with BM MRD ≥0.01%, the majority (n = 120), also received 4 cycles of immunochemotherapy (fludarabine/cyclophosphamide-obinutuzumab). Beyond end of treatment, responses were assessed every 3 month and peripheral blood MRD every 6 months. At median follow-up 36.7 months from treatment start, progression-free and overall survival rates (95% confidence interval) at 3 years were 95.7% (92.0% to 99.5%) and 98% (95.1% to 100%), respectively. Peripheral blood MRD <0.01% rates were 97%, 96%, 90%, 84%, and 89% at months 16, 22, 28, 34, and 40, respectively. No new treatment-related or serious adverse event occurred beyond end of treatment. Thus, in previously untreated, medically fit patients with CLL, a fixed-duration (15 months), MRD-guided approach achieved high survival rates, a persistent MRD benefit beyond the end of treatment, and low long-term toxicity. This trial was registered at www.clinicaltrials.gov as #NCT02666898.

Prevalence of BTK and PLCG2 mutations in a real-life CLL cohort still on ibrutinib after 3 years: a FILO group study.

Mutational analyses performed following acquired ibrutinib resistance have suggested that chronic lymphocytic leukemia (CLL) progression on ibrutinib is linked to mutations in Bruton tyrosine kinase (BTK) and/or phospholipase Cγ2 (PLCG2) genes. Mutational information for patients still on ibrutinib is limited. We report a study aimed to provide a "snapshot" of the prevalence of mutations in a real-life CLL cohort still on ibrutinib after at least 3 years of treatment. Of 204 patients who initiated ibrutinib via an early-access program at 29 French Innovative Leukemia Organization (FILO) centers, 63 (31%) were still on ibrutinib after 3 years and 57 provided a fresh blood sample. Thirty patients had a CLL clone ≥0.5 × 109/L, enabling next-generation sequencing (NGS); BTK and PLCG2 mutations were detected in 57% and 13% of the NGS samples, respectively. After median follow-up of 8.5 months from sample collection, the presence of a BTK mutation was significantly associated with subsequent CLL progression (P = .0005 vs no BTK mutation). Our findings support that mutational analysis should be considered in patients receiving ibrutinib who have residual clonal lymphocytosis, and that clinical trials are needed to evaluate whether patients with a BTK mutation may benefit from an early switch to another treatment.

miR-125b and miR-532-3p predict the efficiency of rituximab-mediated lymphodepletion in chronic lymphocytic leukemia patients. A French Innovative Leukemia Organization study.

The underlying in vivo mechanisms of rituximab action remain incompletely understood in chronic lymphocytic leukemia. Recent data suggest that circulating micro-ribonucleic acids correlate with chronic lymphocytic leukemia progression and response to rituximab. Our study aimed at identifying circulating micro-ribonucleic acids that predict response to rituximab monotherapy in chronic lymphocytic leukemia patients. Using a hierarchical clustering of micro-ribonucleic acid expression profiles discriminating 10 untreated patients with low or high lymphocyte counts, we found 26 micro-ribonucleic acids significantly deregulated. Using individual real-time reverse transcription polymerase chain reaction, the expression levels of micro-ribonucleic acids representative of these two clusters were further validated in a larger cohort (n=61). MiR-125b and miR-532-3p were inversely correlated with rituximab-induced lymphodepletion (P=0.020 and P=0.001, respectively) and with the CD20 expression on CD19+ cells (P=0.0007 and P<0.0001, respectively). In silico analyses of genes putatively targeted by both micro-ribonucleic acids revealed a central role of the interleukin-10 pathway and CD20 (MS4A1) family members. Interestingly, both micro-ribonucleic acids were negatively correlated with MS4A1 expression, while they were positively correlated with MS4A3 and MSA47 Our results identify novel circulating predictive biomarkers for rituximab-mediated lymphodepletion efficacy in chronic lymphocytic leukemia, and suggest a novel molecular mechanism responsible for the rituximab mode of action that bridges miR-125b and miR-532-3p and CD20 family members. (clinicaltrials.gov Identifier: 01370772).

TP53 mutations are early events in chronic lymphocytic leukemia disease progression and precede evolution to complex karyotypes.

TP53 abnormalities lead to resistance to purine analogues and are found in over 40% of patients with refractory chronic lymphocytic leukemia (CLL). At diagnosis, no more than 5% of patients carry the 17p deletion, most cases harbour mutations within the other TP53 allele. The incidence of a TP53 mutation as the only alteration is approximately 5%, but this depends on the sensitivity of the technique. Recently, having a complex karyotype has been considered a strong adverse prognostic factor. However, there are no longitudinal studies simultaneously examining the presence of the 17p deletion, TP53 mutations and karyotype abnormalities. We conducted a retrospective longitudinal study of 31 relapsed/refractory CLL patients. Two to six blood samples per patient were analyzed, with a median follow-up of 8 years. In this report, we assessed the sequence of events of TP53 clonal evolution and correlated the presence of TP53 abnormalities to genetic instability during progression and treatment. Next-generation sequencing allowed the early detection of TP53 mutated clones and was able to be performed on a routine basis, demonstrating an excellent correlation between the Illumina and Ion Torrent technologies. We concluded that TP53 mutations are early events and precede clonal evolution to complex karyotypes. We strongly recommend the early and iterated detection of TP53 mutations in progressive cases.

Old DAT and new data: positive direct antiglobulin test identifies a subgroup with poor outcome among chronic lymphocytic leukemia stage A patients.

Only a minority of chronic lymphocytic leukemia (CLL) patients harboring a positive direct antiglobulin test (DAT) will develop autoimmune hemolytic anemia (AIHA). In a single institution cohort of 378 CLL patients, 56 patients (14.8%) had at least one positive DAT during the course of the disease, either at diagnosis or later. We found no relationship between the time of the first positive DAT and overall survival (OS). However, patients with a positive DAT who did not develop AIHA had the same adverse outcome as patients who developed AIHA. Of the patients who were in Binet stage A at diagnosis, those with a positive DAT had a significantly shorter OS, regardless of their IGHV mutational status, however, there was a strong association with VH1-69. By multivariate analysis, a positive DAT was found to be an independent adverse prognostic factor for OS. Thus, DAT represents a strong adverse prognostic factor and its determination should be repeated during follow-up.

The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia.

B-cell antigen receptor (BCR)-mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium-calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.

Excess mortality after treatment with fludarabine and cyclophosphamide in combination with alemtuzumab in previously untreated patients with chronic lymphocytic leukemia in a randomized phase 3 trial.

A French and Belgian multicenter phase 3 trial was conducted in medically fit patients with untreated chronic lymphocytic leukemia. Of 178 patients enrolled in the study, 165 were randomly assigned to receive 6 courses of oral fludarabine and cyclophosphamide (FC) in combination with rituximab (FCR; 375 mg/m(2) in cycle one, 500 mg/m(2) in all subsequent cycles) or alemtuzumab (FCCam; 30 mg subcutaneously injected on cycle days 1-3); each cycle was 28 days. Recruitment was halted prematurely because of excess toxicity; 8 patients died in the FCCam group, 3 from lymphoma and 5 from in-fection. Overall response rates were 91% with FCR and 90% with FCCam (P = .79). Complete remission rates were 33.75% with FCR and 19.2% with FCCam (P = .04). Three-year progression-free survival was 82.6% with FCR and 72.5% with FCCam (P = .21). Three-year overall survival was similar between the 2 arms at 90.1% in the FCR arm and 86.4% in the FCCam arm (P = .27). These results indicate that the FCCam regimen for the treatment of advanced chronic lymphocytic leukemia was not more effective than the FCR regimen and was associated with an unfavorable safety profile, representing a significant limitation of its use. This study is registered with www.clinicaltrials.gov as number NCT00564512.

Principal component analysis yields results comparable to those of an elaborate Boolean strategy: simplifying the assessment of measurable residual disease in chronic lymphocytic leukemia patients.

INTRODUCTION: Measurable residual disease (MRD) is becoming increasingly important in the chronic lymphocytic leukemia (CLL) context. It is of independent prognostic significance in terms of favorable progression-free and overall survival. The standardized methods used to assess CLL MRD are based on flow cytometry and real-time quantitative PCR. We here present a nine-color assay for CLL MRD with the ROR-1 marker antigen as recommended by the European Research Initiative (ERIC) on CLL; the sensitivity is at least 10-5. MATERIALS AND METHODS: We used 54 samples to develop a new principal component analysis (PCA) method based on the Kaluza© "radar" presentation mode. We used a Navios flow cytometer (Beckman Coulter©). RESULTS: We confirmed the linearity of our method over more than five dilutions. The specificity limit was 1.3×10-6 and the lower limit of detection was 3.6×10-6. Compared to the Boolean method, the sensitivity, specificity, and positive and negative predictive values of our PCA method were 100%. When MRD was detectable, PCA and Boolean assays were in agreement (linear regression, R2 = 0.99). CONCLUSION: We developed a new PCA-based method for detection of CLL MRD. Our method is comparable to that of the consensus method in terms of sensitivity, and it is also much easier and faster.

Immuno-regulatory malignant B cells contribute to Chronic Lymphocytic Leukemia progression.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell neoplasm ranging from indolent to rapidly progressive disease. Leukemic cell subsets with regulatory properties evade immune clearance; however, the contribution of such subsets during CLL progression is not completely elucidated. Here, we report that CLL B cells crosstalk with their immune counterparts, notably by promoting the regulatory T (Treg) cell compartment and shaping several helper T (Th) subsets. Among various constitutively- and BCR/CD40-mediated factors secreted, tumour subsets co-express two important immunoregulatory cytokines, IL10 and TGFß1, both associated with a memory B cell phenotype. Neutralizing secreted IL10 or inhibiting the TGFß signalling pathway demonstrated that these cytokines are mainly involved in Th- and Treg differentiation/maintenance. In line with the regulatory subsets, we also demonstrated that a CLL B cell population expresses FOXP3, a marker of regulatory T cells. Analysis of IL10, TGFß1 and FOXP3 positive subpopulations frequencies in CLL samples discriminated 2 clusters of untreated CLL patients that were significantly different in Tregs frequency and time-to-treatment. Since this distinction was pertinent to disease progression, the regulatory profiling provides a new rationale for patient stratification and sheds light on immune dysfunction in CLL.

A fixed-duration immunochemotherapy approach in CLL: 5.5-year results from the phase 2 ICLL-07 FILO trial.

In previously untreated, medically fit patients with chronic lymphocytic leukemia (CLL), research is focused on developing fixed-duration strategies to improve long-term outcomes while sparing patients from serious toxicities. The ICLL-07 trial evaluated a fixed-duration (15-month) immunochemotherapy approach in which after obinutuzumab-ibrutinib induction for 9 months, patients (n = 10) in complete remission (CR) with bone marrow (BM) measurable residual disease (MRD) <0.01% continued only ibrutinib 420 mg/day for 6 additional months (I arm), whereas the majority (n = 115) received up to 4 cycles of fludarabine/cyclophosphamide-obinutuzumab 1000 mg alongside the ibrutinib (I-FCG arm). Primary analysis at month 16 showed that 84 of 135 (62.2%) patients enrolled achieved CR with a BM MRD <0.01%. Here, we report follow-up at median 63 months. Peripheral blood (PB) MRD was assessed 6 monthly beyond the end of treatment using a highly sensitive (10-6) flow cytometry technique. In the I-FCG arm, the PB MRD <0.01% rate (low-level positive <0.01% or undetectable with limit of detection ≤10-4) in evaluable patients was still 92.5% (74/80) at month 40 and 80.6% (50/62) at month 64. No differences in the PB MRD status were apparent per to the IGHV mutational status. In the overall population, 4-year progression-free and overall survival rates were 95.5% and 96.2%, respectively. Twelve deaths occurred overall. Fourteen serious adverse events occurred beyond the end of treatment. Thus, our fixed-duration immunochemotherapy approach produced deep and sustained PB MRD responses, high survival rates, and low long-term toxicity. A randomized trial is needed to compare our immunochemotherapy approach with a chemotherapy-free strategy. This trial was registered at www.clinicaltrials.gov as #NCT02666898.

Prognosis of Binet stage A chronic lymphocytic leukemia patients: the strength of routine parameters.

Recent developments in the management of chronic lymphocytic leukemia (CLL) patients have made necessary the availability of dependable prognostic factors. We have developed a prognostic index derived from the multivariate analysis of 339 stage A patients at diagnosis, exhaustively studied for classical and recent predictive markers. Only 4 biologic parameters were found to be independent predictors of progression-free survival (PFS): serum thymidine kinase (sTK), lymphocytosis, ß2-microglobulin, and CD38 expression. Two groups were distinguishable: cases with no or 1 risk factor (among whom 85% did not progress after 7 years), and cases with 2 or more factors showing a median PFS of 20 months. Finally, we propose an easy, fast, cost-effective strategy for a trustworthy prognostication in stage A patients, who currently represent more than 80% of the CLL population, allowing physicians to adapt follow-up individually.

miRNA profile at diagnosis predicts treatment outcome in patients with B-chronic lymphocytic leukemia: A FILO study.

During many years, chemo-immunotherapy fludarabine-cyclophosphamide-rituximab (FCR) was the gold standard for first line treatment of medically fit patients with symptomatic B-chronic lymphocytic leukemia (CLL). Over the last decade, targeted biotherapies have revolutionized the treatment of B-CLL patients and almost entirely supplanted FCR. However, no biomarker still exists to predict the complete remission (CR) with undetectable minimal residual disease (uMRD) in bone marrow (BM), which remains the best predictive factor for survival. MicroRNAs represent a class of molecular biomarkers which expression is altered in B-CLL. Our study aimed at identifying before treatment blood miRNAs that predict treatment outcome in previously untreated B-CLL patients (NCT01370772, https://clinicaltrials.gov/ct2/show/NCT01370772). Using hierarchical clustering of miRNA expression profiles discriminating 8 patients who achieved CR with BM uMRD from 8 patients who did not achieve CR and displayed detectable BM MRD, we identified 25 miRNAs differentially expressed before treatment. The expression of 11 miRNAs was further validated on a larger cohort (n=123). Based on the dosage of 5 miRNAs at diagnosis, a decision tree was constructed to predict treatment outcome. We identified 6 groups of patients with a distinct probability of being CR with BM uMRD to FCR treatment, ranging from 72% (miR-125b, miR-15b and miR-181c high) to 4% (miR-125b and miR-193b low). None of the patients displaying high expression levels of miR-125b, miR-15b and miR-181c relapsed during study follow-up. In contrast, patients with low miR-15b and high miR-412, or with low miR-125b and miR-193b, demonstrated significant low PFS. RNA sequencing of blood at diagnosis identified that patients relapsing after treatment are characterized by significant enrichment of gene signatures related to cell cycle, MYC target genes, metabolism and translation regulation. Conversely, patients achieving CR with BM uMRD displayed significant enrichment in genes related to communication between CLL cells and the microenvironment, immune system activation and upregulation of polycomb PRC2 complex target genes. Our results suggest that blood miRNAs are potent predictive biomarkers for FCR treatment efficacy and might be implicated in the FCR efficacy in B-CLL patients, providing new insight into unmet need for the treatment of B-CLL patients and identifying pathways predictive of patients' remission. Clinical trial registration: ClinicalTrials.gov, identifier NCT01370772.

Humoral response to mRNA anti-COVID-19 vaccines BNT162b2 and mRNA-1273 in patients with chronic lymphocytic leukemia.

Immunocompromised individuals such as patients with chronic lymphocytic leukemia (CLL) are at risk of impaired immune responses to vaccination. The objective of our study was to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses in patients with CLL after the first, second, and third doses of the BNT162b2 or mRNA-1273 vaccines and after a single dose for patients with confirmed previous COVID-19. In all, 530 patients were included in the study. Patients received 2 doses at a 4-week interval and a third dose if they were seronegative after the second dose. Response rate was 27% after dose 1 and 52% after dose 2. Post-dose 2 treatment-naïve patients had the highest response rate (72%) followed by patients previously treated by chemoimmunotherapy (60%). Among patients receiving therapy, those receiving Bruton tyrosine kinase inhibitor alone (22%) or in combination with anti-CD20 monoclonal antibodies or venetoclax (0%) had the poorer response rate whereas patients who received venetoclax monotherapy achieved a significantly higher response rate (52%). A multivariable analysis identified age older than 65 years, ongoing CLL treatment, and gamma globulin ≤6 g/L as independent predictors of the absence of seroconversion. Post-dose 2 seronegative patients had a global response rate of 35% after dose 3. This study provides an argument for the use of a third dose and for prophylactic SARS-CoV-2 neutralizing monoclonal antibodies.

Angiogenic factors could help us to define patients obtaining complete response with undetectable minimal residual disease in untreated CLL patients treated by FCR: results from the CLL2010FMP, a FILO study.

Angiogenesis is in a constant balance between pro and anti-angiogenic factors. Neoangiogenesis, implicated in metastatic spreading is characterized in solid cancers, but fairly new in chronic lymphocytic leukemia (CLL). We hypothesize that secretion of angiogenic factors could be correlated to the pathogenesis of CLL, and therefore predict the outcome of patients. We investigated concentrations of 22 cytokines and chemokines in 137 non-del 17p B-CLL patients, treated with a fludarabine-cyclophosphamide-rituximab (FCR)-based regimen. We constructed a biomarker index defining different risk groups based on lymphocyte count, the intensity of CD20 antigen on CD19+ cells, Ang-2, and PDGF-BB plasma concentrations at diagnosis. Four groups were defined, exhibiting specific molecular signatures and correlated with progression-free survival of patients. Our results suggest that we can determine at diagnosis of non-del 17p B-CLL patients, those with a very high probability of progression-free survival, independently of IGVH mutational status and residual disease at the end of treatment.